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1
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Zheng Y, Gu Z, Shudde CE, Piper TL, Wang X, Aleck GA, Zhou J, King D, Chanda MK, Trinch L, Zou W, Courtney AH. An engineered viral protein activates STAT5 to prevent T cell suppression. Sci Immunol 2025; 10:eadn9633. [PMID: 40408430 DOI: 10.1126/sciimmunol.adn9633] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2024] [Revised: 01/08/2025] [Accepted: 04/30/2025] [Indexed: 05/25/2025]
Abstract
T cell therapy efficacy can be compromised if cytokine-induced Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling is dysregulated or insufficient to sustain functionality. Here, we demonstrate that LCK kinase activity can be recruited to noncanonical protein substrates to directly activate targeted STAT proteins in T cells. STAT activation was accomplished by engineering the herpesvirus saimiri tyrosine kinase interacting protein (TIP) to provide a platform for the enforced recruitment of LCK to STAT proteins. We determined that a minimal region of TIP that binds to LCK could be combined with STAT binding sites derived from endogenous cytokine receptors. These constructs activated targeted STAT proteins in a cytokine-independent manner. We identified a STAT5 activator that sustained CD8+ T cell survival and cytotoxic function ex vivo in the absence of interleukin-2. Tumor outgrowth was reduced in vivo because of enhanced T cell persistence and functionality. Single-cell transcriptomics revealed that the STAT5 activator prevented the expression of genes associated with an exhausted T cell fate. Our findings demonstrate that signaling pathways can be rewired in T cells to sustain their function in solid tumors.
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Affiliation(s)
- Yating Zheng
- Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Zehui Gu
- Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Claire E Shudde
- Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Taylor L Piper
- Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Xinyu Wang
- Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Grace A Aleck
- Cellular and Molecular Biology Training Program, University of Michigan, Ann Arbor, MI 48109, USA
| | - Jiajia Zhou
- Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA
| | - Dana King
- BRCF Bioinformatics Core, University of Michigan, Ann Arbor, MI 48109, USA
| | - Monica K Chanda
- Cancer Biology Training Program, University of Michigan, Ann Arbor, MI 48109, USA
| | - Lilliana Trinch
- Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Weiping Zou
- Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA
- Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
- Rogel Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA
| | - Adam H Courtney
- Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA
- Rogel Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA
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2
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Pagano MA, Tibaldi E, Palù G, Brunati AM. Viral proteins and Src family kinases: Mechanisms of pathogenicity from a “liaison dangereuse”. World J Virol 2013; 2:71-78. [PMID: 24175231 PMCID: PMC3785045 DOI: 10.5501/wjv.v2.i2.71] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Revised: 01/07/2013] [Accepted: 01/24/2013] [Indexed: 02/05/2023] Open
Abstract
To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins (VPs) and resident protein partners. Among the latter, Src family kinases (SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cell’s infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology (SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signaling pathways so that viruses can utilize the cell’s machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.
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3
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Kraskouskaya D, Duodu E, Arpin CC, Gunning PT. Progress towards the development of SH2 domain inhibitors. Chem Soc Rev 2013; 42:3337-70. [DOI: 10.1039/c3cs35449k] [Citation(s) in RCA: 83] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
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4
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Isakov N, Altman A. PKC-theta-mediated signal delivery from the TCR/CD28 surface receptors. Front Immunol 2012; 3:273. [PMID: 22936936 PMCID: PMC3425079 DOI: 10.3389/fimmu.2012.00273] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2012] [Accepted: 08/09/2012] [Indexed: 12/23/2022] Open
Abstract
Protein kinase C-theta (PKCθ) is a key enzyme in T lymphocytes, where it plays an important role in signal transduction downstream of the activated T cell antigen receptor (TCR) and the CD28 costimulatory receptor. Interest in PKCθ as a potential drug target has increased following recent findings that PKCθ is essential for harmful inflammatory responses mediated by Th2 (allergies) and Th17 (autoimmunity) cells as well as for graft-versus-host disease (GvHD) and allograft rejection, but is dispensable for beneficial responses such as antiviral immunity and graft-versus-leukemia (GvL) response. TCR/CD28 engagement triggers the translocation of the cytosolic PKCθ to the plasma membrane (PM), where it localizes at the center of the immunological synapse (IS), which forms at the contact site between an antigen-specific T cell and antigen-presenting cells (APC). However, the molecular basis for this unique localization, and whether it is required for its proper function have remained unresolved issues until recently. Our recent study resolved these questions by demonstrating that the unique V3 (hinge) domain of PKCθ and, more specifically, a proline-rich motif within this domain, is essential and sufficient for its localization at the IS, where it is anchored to the cytoplasmic tail of CD28 via an indirect mechanism involving Lck protein tyrosine kinase (PTK) as an intermediate. Importantly, the association of PKCθ with CD28 is essential not only for IS localization, but also for PKCθ-mediated activation of downstream signaling pathways, including the transcription factors NF-κB and NF-AT, which are essential for productive T cell activation. Hence, interference with formation of the PKCθ-Lck-CD28 complex provides a promising basis for the design of novel, clinically useful allosteric PKCθ inhibitors. An additional recent study demonstrated that TCR triggering activates the germinal center kinase (GSK)-like kinase (GLK) and induces its association with the SLP-76 adaptor at the IS, where GLK phosphorylates the activation loop of PKCθ, converting it into an active enzyme. This recent progress, coupled with the need to study the biology of PKCθ in human T cells, is likely to facilitate the development of PKCθ-based therapeutic modalities for T cell-mediated diseases.
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Affiliation(s)
- Noah Isakov
- The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben-Gurion University of the Negev Beer Sheva, Israel
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Mangels C, Frank AO, Ziegler J, Klingenstein R, Schweimer K, Willbold D, Korth C, Rösch P, Schwarzinger S. Binding of TCA to the Prion Protein: Mechanism, Implication for Therapy, and Application as Probe for Complex Formation of Bio-macromolecules. J Biomol Struct Dyn 2012; 27:163-70. [DOI: 10.1080/07391102.2009.10507306] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Affiliation(s)
- Christian Mangels
- a Department of Biopolymers , University of Bayreuth Universitätsstrasse 30 , 95440 , Bayreuth , Germany
| | - Andreas O. Frank
- a Department of Biopolymers , University of Bayreuth Universitätsstrasse 30 , 95440 , Bayreuth , Germany
| | - Jan Ziegler
- a Department of Biopolymers , University of Bayreuth Universitätsstrasse 30 , 95440 , Bayreuth , Germany
| | - Ralph Klingenstein
- b Institute of Neuropathology, Heinrich-Heine-University , Moorenstr. 5, 40225 , Düsseldorf , Germany
| | - Kristian Schweimer
- a Department of Biopolymers , University of Bayreuth Universitätsstrasse 30 , 95440 , Bayreuth , Germany
| | - Dieter Willbold
- c Institute for Physical Biology Heinrich-Heine-University Düsseldorf and Institute of Neuroscience and Biophysics 2 at the Research Centre, Jülich , 52425 , Jülich , Germany
| | - Carsten Korth
- b Institute of Neuropathology, Heinrich-Heine-University , Moorenstr. 5, 40225 , Düsseldorf , Germany
| | - Paul Rösch
- a Department of Biopolymers , University of Bayreuth Universitätsstrasse 30 , 95440 , Bayreuth , Germany
| | - Stephan Schwarzinger
- a Department of Biopolymers , University of Bayreuth Universitätsstrasse 30 , 95440 , Bayreuth , Germany
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6
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Species restriction of Herpesvirus saimiri and Herpesvirus ateles: Human lymphocyte transformation correlates with distinct signaling properties of viral oncoproteins. Virus Res 2012; 165:179-89. [DOI: 10.1016/j.virusres.2012.02.014] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2011] [Revised: 02/06/2012] [Accepted: 02/16/2012] [Indexed: 01/05/2023]
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A molecular model for the differential activation of STAT3 and STAT6 by the herpesviral oncoprotein tip. PLoS One 2012; 7:e34306. [PMID: 22509288 PMCID: PMC3320567 DOI: 10.1371/journal.pone.0034306] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2012] [Accepted: 02/27/2012] [Indexed: 01/27/2023] Open
Abstract
Constitutive STAT signaling provides growth promoting signals in many forms of malignancy. We performed molecular modeling and molecular dynamics studies of the interaction between the regulatory Src homology 2 (SH2) domains of STAT3 and 6 with phosphorylated peptides of the herpesviral oncoprotein Tip, which facilitates Src kinase mediated STAT-activation and T cell proliferation. The studies give insight into the ligand binding specificity of the STAT SH2 domains and provide the first model for the differential activation of STAT3 or STAT6 by two distinct regions of the viral Tip protein. The biological relevance of the modeled interactions was then confirmed by activation studies using corresponding recombinant oncoproteins, and finally by respective recombinant viruses. The functional data give experimental validation of the molecular dynamics study, and provide evidence for the involvement of STAT6 in the herpesvirus induced T cell proliferation.
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Identification of the variant Ala335Val of MED25 as responsible for CMT2B2: molecular data, functional studies of the SH3 recognition motif and correlation between wild-type MED25 and PMP22 RNA levels in CMT1A animal models. Neurogenetics 2009; 10:275-87. [PMID: 19290556 PMCID: PMC2847151 DOI: 10.1007/s10048-009-0183-3] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2008] [Accepted: 02/19/2009] [Indexed: 01/30/2023]
Abstract
Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous disorder. All mendelian patterns of inheritance have been described. We identified a homozygous p.A335V mutation in the MED25 gene in an extended Costa Rican family with autosomal recessively inherited Charcot-Marie-Tooth neuropathy linked to the CMT2B2 locus in chromosome 19q13.3. MED25, also known as ARC92 and ACID1, is a subunit of the human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator. MED25 was identified by virtue of functional association with the activator domains of multiple cellular and viral transcriptional activators. Its exact physiological function in transcriptional regulation remains obscure. The CMT2B2-associated missense amino acid substitution p.A335V is located in a proline-rich region with high affinity for SH3 domains of the Abelson type. The mutation causes a decrease in binding specificity leading to the recognition of a broader range of SH3 domain proteins. Furthermore, Med25 is coordinately expressed with Pmp22 gene dosage and expression in transgenic mice and rats. These results suggest a potential role of this protein in the molecular etiology of CMT2B2 and suggest a potential, more general role of MED25 in gene dosage sensitive peripheral neuropathy pathogenesis.
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9
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Solheim SA, Petsalaki E, Stokka AJ, Russell RB, Taskén K, Berge T. Interactions between the Fyn SH3-domain and adaptor protein Cbp/PAG derived ligands, effects on kinase activity and affinity. FEBS J 2008; 275:4863-74. [PMID: 18721137 DOI: 10.1111/j.1742-4658.2008.06626.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched domains is a transmembrane adaptor protein primarily involved in negative regulation of T-cell activation by recruitment of C-terminal Src kinase (Csk), a protein tyrosine kinase which represses Src kinase activity through C-terminal phosphorylation. Recruitment of Csk occurs via SH2-domain binding to PAG pTyr317, thus, the interaction is highly dependent on phosphorylation performed by the Src family kinase Fyn, which docks onto PAG using a dual-domain binding mode involving both SH3- and SH2-domains of Fyn. In this study, we investigated Fyn SH3-domain binding to 14-mer peptide ligands derived from Cbp/PAG-enriched microdomains sequence using biochemical, biophysical and computational techniques. Interaction kinetics and dissociation constants for the various ligands were determined by SPR. The local structural impact of ligand association has been evaluated using CD, and molecular modelling has been employed to investigate details of the interactions. We show that data from these investigations correlate with functional effects of ligand binding, assessed experimentally by kinase assays using full-length PAG proteins as substrates. The presented data demonstrate a potential method for modulation of Src family kinase tyrosine phosphorylation through minor changes of the substrate SH3-interacting motif.
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Affiliation(s)
- Silje A Solheim
- The Biotechnology Centre of Oslo, University of Oslo, Norway
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10
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Wittlich M, Wiesehan K, Koenig BW, Willbold D. Expression, purification, and membrane reconstitution of a CD4 fragment comprising the transmembrane and cytoplasmic domains of the receptor. Protein Expr Purif 2007; 55:198-207. [PMID: 17613246 DOI: 10.1016/j.pep.2007.05.007] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2007] [Revised: 05/08/2007] [Accepted: 05/21/2007] [Indexed: 10/23/2022]
Abstract
The transmembrane glycoprotein CD4 plays a prominent role in the adaptive immune response. CD4 is displayed primarily on the surface of T helper cells, but also on subsets of memory and regulatory T lymphocytes, macrophages, and dendritic cells. Binding of the lymphocyte specific tyrosine kinase p56(lck) to the cytoplasmic domain of CD4 is crucial for antigen receptor-mediated signal transduction. The human immunodeficiency virus (HIV) utilizes CD4 as the main receptor for T cell invasion. The virus has developed multiple strategies for down-regulation of CD4 in infected cells. Physical interactions of viral proteins VpU and Nef with the cytoplasmic tail of CD4 initiate a cascade of events leading to degradation of CD4. Here we report heterologous expression and purification of a CD4 fragment comprising the transmembrane and cytoplasmic domains of human CD4. A synthetic gene encoding CD4 amino acid residues 372-433 and a protease cleavage site was cloned into the pTKK19xb/ub plasmid. The CD4 fragment was expressed in Escherichia coli C43(DE3) cells as a ubiquitin fusion with an N-terminal His-tag, isolated, released by PreScission proteolytic cleavage, and purified to homogeneity. Incorporation of the recombinant CD4 fragment in lipid membranes and physical interaction with the cytoplasmic domain of VpU was demonstrated by centrifugation assays followed by reversed phase chromatographic analysis of the composition of the proteoliposomes. A high resolution NMR spectrum of uniformly (15)N-labeled CD4 peptide in membrane simulating micelles proves the possibility of solution NMR studies of this CD4 fragment and of its molecular complexes.
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Affiliation(s)
- Marc Wittlich
- Institut für Physikalische Biologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany
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11
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Bauer F, Sticht H. A proline to glycine mutation in the Lck SH3-domain affects conformational sampling and increases ligand binding affinity. FEBS Lett 2007; 581:1555-60. [PMID: 17382937 DOI: 10.1016/j.febslet.2007.03.012] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2007] [Revised: 03/01/2007] [Accepted: 03/06/2007] [Indexed: 11/23/2022]
Abstract
Loop flexibility is discussed as a factor that affects ligand binding affinity of SH3 domains. To test this hypothesis, we designed a mutant in which a proline in the RT-loop of the human Lck SH3-domain is replaced by glycine. The dynamics and ligand binding properties of wild-type and mutant LckSH3 were studied by fluorescence and NMR spectroscopy as well as molecular dynamics simulations. Although the mutated residue does not form direct contacts with the ligand, the mutation increases ligand affinity by a factor of eight. The mutant exhibits increased loop flexibility and enhanced sampling of binding-competent conformations. This effect is expected to facilitate ligand binding itself and might also allow formation of tighter contacts in the complex thus resulting in an increased binding affinity.
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Affiliation(s)
- Finn Bauer
- Abteilung Bioinformatik, Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany
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12
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Mitchell JL, Trible RP, Emert-Sedlak LA, Weis DD, Lerner EC, Applen JJ, Sefton BM, Smithgall TE, Engen JR. Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein. J Mol Biol 2007; 366:1282-93. [PMID: 17207813 PMCID: PMC2262936 DOI: 10.1016/j.jmb.2006.12.026] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2006] [Revised: 12/06/2006] [Accepted: 12/07/2006] [Indexed: 11/15/2022]
Abstract
Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific member of the Src family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen-exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured, so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel-filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of activating Lck kinase activity strongly and was multiply phosphorylated by Lck. Hydrogen-exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogen atoms became deuterated after only 10 s of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium-labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that, although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.
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Affiliation(s)
| | - Ronald P. Trible
- Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Lori A. Emert-Sedlak
- Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - David D. Weis
- Department of Chemistry, University of New Mexico, Albuquerque, NM 87131
| | - Edwina C. Lerner
- Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Jeremy J. Applen
- Department of Chemistry, University of New Mexico, Albuquerque, NM 87131
| | | | - Thomas E. Smithgall
- Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - John R. Engen
- Department of Chemistry, University of New Mexico, Albuquerque, NM 87131
- Chemistry & Chemical Biology and The Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA 02115
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13
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Weis DD, Kjellen P, Sefton BM, Engen JR. Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri Tip. Protein Sci 2007; 15:2402-10. [PMID: 17008721 PMCID: PMC2242400 DOI: 10.1110/ps.052016406] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
The Tip protein from Herpesvirus saimiri interacts with the SH3 domain from the Src-family kinase Lck via a proline-containing sequence termed LBD1. Src-family kinase SH3 domains related to Lck have been shown to be dynamic in solution and partially unfold under physiological conditions. The rate of such partial unfolding is reduced by viral protein binding. To determine if the Lck SH3 domain displayed similar behavior, the domain was investigated with hydrogen exchange and mass spectrometry. Lck SH3 was found to be highly dynamic in solution. While other SH3 domains require as much as 10,000 sec to become totally deuterated, Lck SH3 became almost completely labeled within 200 sec. A partial unfolding event involving 8-10 residues was observed with a half-life of approximately 10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved.
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Affiliation(s)
- David D Weis
- Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA
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14
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Heck E, Friedrich U, Gack MU, Lengenfelder D, Schmidt M, Müller-Fleckenstein I, Fleckenstein B, Ensser A, Biesinger B. Growth transformation of human T cells by herpesvirus saimiri requires multiple Tip-Lck interaction motifs. J Virol 2006; 80:9934-42. [PMID: 17005671 PMCID: PMC1617286 DOI: 10.1128/jvi.01112-06] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.
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Affiliation(s)
- Elke Heck
- Institut für Klinische und Molekulare Virologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schlossgarten 4, D-91054 Erlangen, Germany
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15
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Romir J, Lilie H, Egerer-Sieber C, Bauer F, Sticht H, Muller YA. Crystal structure analysis and solution studies of human Lck-SH3; zinc-induced homodimerization competes with the binding of proline-rich motifs. J Mol Biol 2006; 365:1417-28. [PMID: 17118402 DOI: 10.1016/j.jmb.2006.10.058] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2006] [Revised: 10/12/2006] [Accepted: 10/16/2006] [Indexed: 01/24/2023]
Abstract
In cytosolic Src-type tyrosine kinases the Src-type homology 3 (SH3) domain binds to an internal proline-rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src-type kinase Lck plays an important role during T-cell activation and development, since it phosphorylates the T-cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near-atomic resolution of 1.0 A. Unexpectedly, the Lck-SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc-binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline-rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck-SH3 domain also forms dimers in solution. The Zn(2+) dissociation constant from the Lck-SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline-rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc-induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck-SH3 homodimerization and binding of regulatory proline-rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc-binding site are highly conserved among Lck orthologues but not in other Src-type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T-cell activation.
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Affiliation(s)
- Johannes Romir
- Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany
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16
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Brinkmann MM, Schulz TF. Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae. J Gen Virol 2006; 87:1047-1074. [PMID: 16603506 DOI: 10.1099/vir.0.81598-0] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
The human gamma(1)-herpesvirus Epstein-Barr virus (EBV) and the gamma(2)-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV), rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) all contain genes located adjacent to the terminal-repeat region of their genomes, encoding membrane proteins involved in signal transduction. Designated 'terminal membrane proteins' (TMPs) because of their localization in the viral genome, they interact with a variety of cellular signalling molecules, such as non-receptor protein tyrosine kinases, tumour-necrosis factor receptor-associated factors, Ras and Janus kinase (JAK), thereby initiating further downstream signalling cascades, such as the MAPK, PI3K/Akt, NF-kappaB and JAK/STAT pathways. In the case of TMPs expressed during latent persistence of EBV and HVS (LMP1, LMP2A, Stp and Tip), their modulation of intracellular signalling pathways has been linked to the provision of survival signals to latently infected cells and, hence, a contribution to occasional cellular transformation. In contrast, activation of similar pathways by TMPs of KSHV (K1 and K15) and RRV (R1), expressed during lytic replication, may extend the lifespan of virus-producing cells, alter their migration and/or modulate antiviral immune responses. Whether R1 and K1 contribute to the oncogenic properties of KSHV and RRV has not been established satisfactorily, despite their transforming qualities in experimental settings.
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Affiliation(s)
- Melanie M Brinkmann
- Institut für Virologie, Medizinische Hochschule Hannover, Carl-Neuberg Str. 1, D-30625 Hannover, Germany
| | - Thomas F Schulz
- Institut für Virologie, Medizinische Hochschule Hannover, Carl-Neuberg Str. 1, D-30625 Hannover, Germany
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Heinemann S, Biesinger B, Fleckenstein B, Albrecht JC. NFkappaB signaling is induced by the oncoprotein Tio through direct interaction with TRAF6. J Biol Chem 2006; 281:8565-72. [PMID: 16452479 DOI: 10.1074/jbc.m510891200] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
The transcription factor NFkappaB is a major regulator of genes involved in inflammation and oncogenesis. NFkappaB is induced upon stimulation of cellular receptors coupled to different intracellular signaling molecules. Further downstream, TRAF6 links at least two receptor pathways to take control of IkappaB, the administrator of NFkappaB activity. Here we report on a strong NFkappaB activation by Tio, a unique herpesviral oncoprotein promoting transformation of human T cells in a Src-kinase-dependent manner. NFkappaB induction by Tio is independent of Src-kinase interaction and tyrosine phosphorylation of Tio. Mutation of a glutamic acid-rich motif at the N terminus of Tio, corresponding to a TRAF6 consensus binding motif, completely abrogated NFkappaB activation. Cotransfection of a dominant negative TRAF6 construct led to a decrease in NFkappaB activation. Furthermore, we provide evidence that TRAF6 directly binds to the Tio oncoprotein. Identification of TRAF6 as the direct target of Tio describes a novel mechanism for the constitutive activation of NFkappaB through an oncoprotein.
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Affiliation(s)
- Stefanie Heinemann
- Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany.
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Deng L, Velikovsky CA, Swaminathan CP, Cho S, Mariuzza RA, Huber R. Structural basis for recognition of the T cell adaptor protein SLP-76 by the SH3 domain of phospholipase Cgamma1. J Mol Biol 2005; 352:1-10. [PMID: 16061254 PMCID: PMC2753203 DOI: 10.1016/j.jmb.2005.06.072] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2005] [Revised: 06/24/2005] [Accepted: 06/30/2005] [Indexed: 11/29/2022]
Abstract
The enzyme phospholipase Cgamma1 (PLCgamma1) is essential for T cell signaling and activation. Following T cell receptor ligation, PLCgamma1 interacts through its SH2 and SH3 domains with the adaptors LAT and SLP-76, respectively, to form a multiprotein signaling complex that leads to activation of PLCgamma1 by Syk tyrosine kinases. To identify the binding site for PLCgamma1 in SLP-76, we used isothermal titration calorimetry to measure affinities for the interaction of PLCgamma1-SH3 with a set of overlapping peptides spanning the central proline-rich region of SLP-76. PLCgamma1-SH3 bound with high specificity to the SLP-76 motif 186PPVPPQRP193, which represents the minimal binding site. To understand the basis for selective recognition, we determined the crystal structures of PLCgamma1-SH3 in free form, and bound to a 10-mer peptide containing this site, to resolutions of 1.60 A and 1.81 A, respectively. The structures reveal that several key contacting residues of the SH3 shift toward the SLP-76 peptide upon complex formation, optimizing the fit and strengthening hydrophobic interactions. Selectivity results mainly from strict shape complementarity between protein and peptide, rather than sequence-specific hydrogen bonding. In addition, Pro193 of SLP-76 assists in positioning Arg192 into the compass pocket of PLCgamma1-SH3, which coordinates the compass residue through an unusual aspartate. The PLCgamma1-SH3/SLP-76 structure provides insights into ligand binding by SH3 domains related to PLCgamma1-SH3, as well as into recognition by PLCgamma1 of signaling partners other than SLP-76.
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Affiliation(s)
| | | | | | | | - Roy A. Mariuzza
- Corresponding author E-mail address of the corresponding author:
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Bauer F, Schweimer K, Meiselbach H, Hoffmann S, Rösch P, Sticht H. Structural characterization of Lyn-SH3 domain in complex with a herpesviral protein reveals an extended recognition motif that enhances binding affinity. Protein Sci 2005; 14:2487-98. [PMID: 16155203 PMCID: PMC2253286 DOI: 10.1110/ps.051563605] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
The Src homology 3 (SH3) domain of the Src family kinase Lyn binds to the herpesviral tyrosine kinase interacting protein (Tip) more than one order of magnitude stronger than other closely related members of the Src family. In order to identify the molecular basis for high-affinity binding, the structure of free and Tip-bound Lyn-SH3 was determined by NMR spectroscopy. Tip forms additional contacts outside its classical proline-rich recognition motif and, in particular, a strictly conserved leucine (L186) of the C-terminally adjacent sequence stretch packs into a hydrophobic pocket on the Lyn surface. Although the existence of this pocket is no unique property of Lyn-SH3, Lyn is the only Src family kinase that contains an additional aromatic residue (H41) in the n-Src loop as part of this pocket. H41 covers L186 of Tip by forming tight hydrophobic contacts, and model calculations suggest that the increase in binding affinity compared with other SH3 domains can mainly be attributed to these additional interactions. These findings indicate that this pocket can mediate specificity even between otherwise closely related SH3 domains.
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Affiliation(s)
- Finn Bauer
- Lehrstuhl für Biopolymere, Universität Bayreuth, 95440 Bayreuth, Germany
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Albrecht JC, Müller-Fleckenstein I, Schmidt M, Fleckenstein B, Biesinger B. Tyrosine phosphorylation of the Tio oncoprotein is essential for transformation of primary human T cells. J Virol 2005; 79:10507-13. [PMID: 16051843 PMCID: PMC1182665 DOI: 10.1128/jvi.79.16.10507-10513.2005] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human T cells are transformed to antigen-independent permanent growth in vitro upon infection with herpesvirus saimiri subgroup C strains. The viral oncoproteins required for this process, StpC and Tip, could be replaced by Tio, the oncoprotein of herpesvirus ateles. Here we demonstrate that proliferation of lymphocytes transformed with Tio-recombinant herpesvirus saimiri required the activity of Src family kinases. Src kinases had previously been identified as interaction partners of Tio. This interaction was now shown to be independent of any of the four tyrosine residues of Tio but to be dependent on an SH3-binding motif. Mutations within this motif abrogated the transforming capabilities of Tio-recombinant herpesvirus saimiri. Furthermore, kinase interaction resulted in the phosphorylation of Tio on a single tyrosine residue at position 136. Mutation of this residue in the viral context revealed that this phosphorylation site, but none of the other tyrosine residues, was required for T-cell transformation. These data indicate that the interaction of Tio with a Src kinase is essential for both the initiation and the maintenance of T-cell transformation by recombinant herpesvirus saimiri. The requirement for the tyrosine phosphorylation site at position 136 suggests a role for Tio beyond simple deregulation of the kinase.
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Affiliation(s)
- Jens-Christian Albrecht
- Institut für Klinische und Molekulare Virologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schlossgarten 4, D-91054 Erlangen, Germany.
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Hofinger E, Sticht H. Multiple modes of interaction between Lck and CD28. THE JOURNAL OF IMMUNOLOGY 2005; 174:3839-40; author reply 3840. [PMID: 15778335 DOI: 10.4049/jimmunol.174.7.3839-a] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
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Heck E, Lengenfelder D, Schmidt M, Müller-Fleckenstein I, Fleckenstein B, Biesinger B, Ensser A. T-cell growth transformation by herpesvirus saimiri is independent of STAT3 activation. J Virol 2005; 79:5713-20. [PMID: 15827186 PMCID: PMC1082769 DOI: 10.1128/jvi.79.9.5713-5720.2005] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Herpesvirus saimiri (saimirine herpesvirus 2) (HVS), a T-lymphotropic tumor virus, induces lymphoproliferative disease in several species of New World primates. In addition, strains of HVS subgroup C are able to transform T cells of Old World primates, including humans, to permanently growing T-cell lines. In concert with the Stp oncoprotein, the tyrosine kinase-interacting protein (Tip) of HVS C488 is required for T-cell transformation in vitro and lymphoma induction in vivo. Tip was previously shown to interact with the protein tyrosine kinase Lck. Constitutive activation of signal transducers and activators of transcription (STATs) has been associated with oncogenesis and has also been detected in HVS-transformed T-cell lines. Furthermore, Tip contains a putative consensus YXPQ binding motif for the SH2 (src homology 2) domains of STAT1 and STAT3. Tip tyrosine phosphorylation at this site was required for binding of STATs and induction of STAT-dependent transcription. Here we sought to address the relevance of STAT activation for transformation of human T cells by introducing a tyrosine-to-phenylalanine mutation in the YXPQ motif of Tip of HVS C488. Unexpectedly, the recombinant virus was still able to transform human T lymphocytes, but it had lost its capability to activate STAT3 as well as STAT1. This demonstrates that growth transformation by HVS is independent of STAT3 activation.
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Affiliation(s)
- Elke Heck
- Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Schlossgarten 4, D-91054 Erlangen, Germany
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